1. Field of the Invention
The present invention relates to recombinant albumins fused with poly-cysteine peptide and methods for preparing the same.
2. Description of the Related Art
The usability of albumin, as an effective drug in vivo, has been widely known. In particular, in relation to such disease as cancer and rheumatoid arthritis, albumin has been well recognized in its functions as a multi-drug deliverer because of its capability and easiness in improving physical and chemical characteristics such as high accumulation in a target tissue, low invasiveness in a normal tissue, low toxicity, easiness in conjugation with variety of drugs, drug-release in a target tissue, advantageous bio-degradation time, solubility of the fused drug, etc.
In cancer tissues, albumin is preferably metabolized to produce nitrogen and energy. In angiogenetic blood vessels of cancer tissues, EPR effect (enhanced permeability and retention effect) is observed, that is albumin is accumulated selectively in cancer tissues owing to the increased permeability. Abnormality of lymphatic system in cancer tissues accelerates the accumulation of albumin in cancer tissues. So, drug delivery by albumin is an important target of new drug development, which draws our attention world widely.
Chemotherapy, one of the conventional methods treating cancer, is limited in clinical application because of biophysicochemical properties of a drug used. That is, if it is a hydrophobic drug insoluble in water, a drug deliverer has to be prepared with an organic solvent such as polyethoxylated castor oil (CrEL) or polysorbate 80. In that case, not only the drug itself but also the drug deliverer can cause toxicity, which can cause hypersensitivity and other serious neuronal disease. Therefore, an additional medical treatment is required to control such toxicity. That is, when a drug deliverer depends on such organic solvent, there might be a problem of dose-limiting because of toxicity of the drug deliverer. Precisely, dose of a drug for in vivo injection can be limited.
In in vivo drug delivery, in vivo biostability of a drug is also an important factor. Therefore, biostability and half-life can be regulated by the choice of an efficient drug deliverer.
To reduce the toxicity of a drug deliverer and to improve in vivo biostability of the drug itself, it has been continuously attempted to use albumin protein as a drug deliverer.
As an attempt to reduce toxicity of a drug deliverer, methotrexate-albumin conjugate, albumin-binding prodrug of doxorubicin (DOXO-EMCH), and paclitaxel albumin-stabilized nanoparticle formulation (brand name “Abraxane™”) have been clinically tested and acknowledged as effective albumin based drug deliverers.
Abraxane has been approved by FDA, USA, in January, 2005, targeting metastatic breast cancer recurrent patients. It demonstrated excellent capability of drug delivery using albumin. American Bioscience Inc. has developed nab-technology, which is the technology to produce nanoparticles based on albumin, resulting in the success of enveloping a lipid-soluble drug in a nanoparticle. To form albumin nano-particles in the size of 100-200 nm, a target drug is added to albumin in aqueous solution and then jet stream is blown through under the high pressure. By this method, the anticancer agent Paclitaxel is sealed in nano particles, resulting in the product named Abraxane™ which is clinically treated to metastatic breast cancer patients. Compared with the single-treatment of Paclitaxel, the treatment of Abraxane™ demonstrated less side effects such as decrease of leucocytes, mental and body weakness, infection, and muscle ache and the progress of disease was also retarded by the treatment.
The treatment of Methotrexate known as the agent effective in rheumatoid arthritis is based on the mechanism that albumin is over-accumulated in the region of rheumatoid arthritis and metabolized therein. In an animal model, when Methotrexate/albumin deliverer was administered, treatment effect was improved, compared with when Methotrexate alone was treated.
As a method to improve biostability of a drug itself, the drug is conjugated with albumin to improve pharmacokinetic profile. For example, there are Albumin Fusion Technology (Human Genome Sciences), Drug Affinity Complex (DAM™, ConjuChem. Inc.), Performed Conjugated-Drug Affinity complex technology platform (PC-DAM™, ConjuChem. Inc.) and the method provided by Novo Nordisk to conjugate a fatty acid derivative physically to albumin circulating in human body.
Albumin fusion technology provided by Human Genome Sciences is that albumin is conjugated with cytokine protein such as interferon or interleukin by using genetic recombination technique. In this case, protein pegylation like effect is observed along with increase of in vivo half-life, decrease of degradation by protease, and decrease of immune response.
ConjuChem. Inc. has the technique that is able to conjugate a drug with the 34th cysteine free from disulfide bond in albumin by using a reaction group such as N-hydroxysuccinimide ester, isocyanate, maleimide or salicylate group. The company has been conjugated various peptide drugs with albumin by the said method. As an example, CJC-1134-PC, an exendin-4 albumin complex, has been developed as a therapeutic agent for type II diabetes.
Concerning the said drug delivery system, methods of drug conjugation with albumin are largely divided into two categories; which are the method using the 34th cysteine residue and the method using lysine residues on the surface of albumin.
In all the commercial albumins, the 34th cysteine residue of albumin is blocked by sulfhydryl compound such as cysteine, homocysteine and glutathione. So, only 20-60% of the 34th residue can be conjugated with a drug. Therefore, for the effective drug conjugation, it is important to free the 34th cysteine from blocking in commercial albumin.
A method to produce a prodrug has been attempted by targeting the 34th cysteine residue of albumin circulating in vivo. (4-maleimidophenylacetyl) hydrazone derivative and (6-maleimidophenylacetyl) hydrazone derivative of doxorubicin (DOXO-EMCH) are conjugated with the 34th cysteine residue of albumin circulating in vivo within just a few minutes. In that case, the albumin conjugate DOXO-EMCH demonstrates the maximum tolerated dose 4.5 times as high as that of doxorubicin itself that is the albumin conjugate is much effective in treatment than doxorubicin single treatment.
However, even in the case that a drug is conjugated with albumin circulating in vivo, drug binding efficiency is still not high enough because about 70% of those circulating albumin contain free 34th cysteine residue, which is in the form of mercaptalbumin opened for drug conjugation.
Methotrexate-albumin conjugate (MTX-HSA) evaluated until clinical test stage I/II is the example of the second method using lysine residues on the surface of albumin for drug conjugation. This conjugate is produced by direct binding of methotrexate with lysine residue of albumin. However, this methotrexate-albumin conjugate has a problem in identification of the conjugate chemically because of irregularity of numbers of drugs being able to be combined with albumin. Clinical evaluation of the methotrexate-albumin conjugate, a kind of prodrug, is postponed because of its uncertainty in cleavage rate and cleavage product.
According to the method using lysine residue on the surface of albumin, the number of drugs that are conjugated with each albumin is not consistent, so that the albumin-drug conjugate cannot be chemically identified and at the same time the number of drugs conjugated thereto cannot be controlled.
Therefore, to overcome the said disadvantages of the conventional albumin-drug conjugation methods, more effective or improved methods for albumin-drug conjugation, for increasing the number of drugs conjugated to each albumin and for regulating the number of drugs conjugated to each albumin are required.
The present inventors have studied to overcome side effects of the conventional drug delivery system using albumin. As a result, the inventors developed a recombinant albumin by combining albumin with poly-cysteine peptide harboring multi-drug binding sites based on genetic recombination technique. The developed recombinant albumin has the increased number of cysteines that can be useful for drug binding at N-terminal and C-terminal along with the 34th residue, so that it not only improves albumin-drug conjugation efficiency for more effective delivery of a large amount of drugs to a target tissue but also facilitates regulation of the number of fused cysteines to control the number of drugs conjugated to each albumin, indicating that this novel recombinant albumin can be used as an excellent drug deliverer with less side effects than the conventional albumin deliverers. Further, the present inventors completed this invention by confirming that the recombinant albumin developed by the inventors can be effectively used for the non-invasive real-time diagnosis and treatment of disease by combining with a fluorescent material or a contrast agent for molecular imaging.